Book Review: The Second Creation – The age of biological control by the scientists who cloned Dolly

'The Second Creation' explores the history and science of cloning, culminating in the birth of the famous sheep Dolly in 1996 at the Roslin Institute near Edinburgh (see BioNews 1355 and 1131). The book is based on Colin Tudge's interviews with the two researchers who were most prominently involved in cloning Dolly, namely Professor Sir Ian Wilmut and Professor Keith Campbell.

The term 'cloning' can describe many different processes, including asexual reproduction, twinning ('natural cloning') or – as in the case of Dolly – the deliberate replacement of a cell's genetic material with DNA from another source. This last process is known as cloning by nuclear transfer.

The process seems remarkably simple on paper. A cell consists of two main compartments – the nucleus (which contains most of the genetic material) and the cytoplasm (the rest of the cell). To make Dolly, the nucleus was removed from a sheep's egg cell. The resulting enucleated egg was then fused with a sheep's mammary gland cell, which provided the missing nucleus. However, the process is much more complicated in practice than this might sound, as illustrated by the fact that Dolly was the only live lamb born in her cohort (out of 277 embryos cloned).

Appropriately, most of 'The Second Creation' is dedicated to describing previous scientific advances that enabled this achievement. These included the use of a chemical compound to soften the cytoplasm and facilitate enucleation of the egg, the development of a method of using electrical pulses to fuse the enucleated egg with the nuclear donor cell, and the encasing of cloned embryos into agar (jelly-like substance) for brief incubation (about six days) in the oviducts of temporary recipient sheep.

Such incubation was necessary, because in vitro culture of sheep embryos had not yet been optimised. Of the hundreds of cloned embryos, only about ten percent developed successfully to the morula or blastocyst stages, at which they could be considered for transfer to a surrogate. To avoid the unnecessary use of hundreds of sheep as surrogates, the cloned embryos were therefore surgically retrieved from the temporary recipients, and only those that had developed to morula or blastocyst stages were then transferred to surrogates. This was a convoluted process, and the authors acknowledge that 'the story may seem a bit messy – but that's because life is messy; and science is a slice of life.'

Dolly was not the first cloned animal, or even the first cloned mammal. Her significance lies in the use of a specialised cell from an adult animal as the nuclear donor. Prior to Dolly's birth, many doubted that this would be possible, because of a widespread notion that as cells specialise to perform a particular function – for example, milk production in the mammary gland – they can no longer be 'reprogrammed' into an embryonic state.

Cloning at Roslin, and at the collaborating biotech company PPL Therapeutics, was not performed for the sole purpose of answering this fundamental question in biology. Rather, the main driver was commercial interest in genetically engineering livestock to produce useful human proteins (such as blood clotting factors), and then secrete these proteins in their milk, whereupon they could be easily collected and purified.

Previously, embryos had been genetically engineered by microinjecting DNA into them directly following fertilisation. This process was very inefficient, because the microinjected genes integrated into the DNA of only a minority of the embryos. Professor Wilmut pushed to optimise this process, by first introducing the desired genes into cells that were grown in vitro on culture dishes.

Such cells are easier to transform than embryos – partly because there are many more of them, and partly because they can be selected on the basis of whether or not they have taken up the gene(s) of interest. Transforming cells in this way also makes it possible to remove or mutate genes, whereas microinjection allows only for gene addition.

Cells that have been successfully transformed can be identified and then used to perform cloning by nuclear transfer. Professor Campbell improved the process even further, by noticing the importance of using nuclear and cytoplasmic donor cells at a specific stage in their cell cycle (that is, at a particular stage in their cycle of growing, duplicating their DNA, and dividing).

The concluding chapters discuss potential applications of cloning beyond genetic engineering. These include animal conservation, reproduction of elite animals, producing genetically uniform strains of animals for research (without inbreeding), and obtaining embryonic stem cells that are genetically identical to the patients whom these cells are used to treat.

The authors state clearly that they oppose the cloning of humans for several reasons. These reasons include the psychological wellbeing of the people who would be involved, the high levels of prenatal mortality that they observed in the cloned sheep, and the fact that the health implications of cloning are not well understood. The authors also emphasise that cloning by nuclear transfer would not produce an exact replica of a human, because some of our DNA resides in the cytoplasm of our cells and also because our environment plays such a big role in shaping our lives.

Thirty years on from Dolly, much of the science described in the book still holds true. Biology is still very much an experimental science, and embryology requires a high level of manual dexterity. The culture media that we use to grow cells still contain many components with cabalistic qualities that we don't fully understand.

As the book's authors predicted, cloning has been used to reproduce pets and elite animals, including cattle and racehorses, and has also been used in efforts to preserve endangered species (see BioNews 1223 and 935). To date, however, no extinct species have been brought back through cloning, and there have been no confirmed cases of (unnaturally) cloned humans.

One development not predicted by the book has been the rise of genome editing methods. These enable efficient and precise DNA changes to be made directly in embryos, bypassing the steps of cell transformation in culture and cloning by nuclear transfer.

Overall, I enjoyed reading 'The Second Creation' and I appreciated the detailed and accurate description of biological concepts and experimental methods. I found the book somewhat repetitive, but at least this helped me to keep track of the complex processes discussed. I would recommend it to anyone curious about why, and precisely how, Dolly 'and the rest' were cloned.